EP2899281A1

EP2899281A1 - Inhibitors of sterol metabolism for their use to accumulate triglycerides in microalgae, and methods thereof

Info

Publication number: EP2899281A1
Application number: EP14305111.8A
Authority: EP
Inventors: Melissa Conte; Lina-Juana Dolch; Coline Mei; Caroline Barette; Dimitris Petroutsos; Denis Falconet; Juliette Jouhet; Fabrice REBEILLE; Jean-Christophe Cintrat; Eric Marechal
Original assignee: Commissariat a lEnergie Atomique CEA; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Current assignee: Centre National de la Recherche Scientifique CNRS; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2014-01-27
Filing date: 2014-01-27
Publication date: 2015-07-29
Anticipated expiration: 2034-01-27
Also published as: CA2937733A1; WO2015111029A1; EP2899281B1; CN106488985A; CN106488985B; CA2937733C; US10844411B2; US20160348138A1

Abstract

The invention relates to a method for accumulating triacylglycerols in microalgae by inhibiting the sterol metabolism. The invention also relates to a method for producing fatty acids, biofuels, pharmaceutical or cosmetic compositions, and also food supplements, comprising a triacylglycerols accumulation step in microalgae according to the invention. Finally, the invention concerns the use of an inhibitor of sterol metabolism to accumulate triglycerides in microorganisms, and preferably microalgae.

Description

It is acknowledged that oilseed production from crops cannot be diverted from nutritional purpose (Durrett et al., The Plant Journal (2008) 54, 593-607). Therefore, efforts are directed towards the oil production in other organisms like algae (Chisti, Biotechnology Advances 25 (2007) 294-306; Chisti, Trends in Biotechnology, 2008, Vol 26, No. 3; Dismukes et al., Current Opinion in Biotechnology 2008, 19:235-240; Scott et al., Current Opinion in Biotechnology 2010, 21:277-286; Singh et al., Bioresource Technology 102 (2011) 26-34). Studies undergone on triacylglycerol (TAG, also called oil) production in algae (Table 1 below) have focused on the increase of TAG in cytosolic droplets.

Table 1: Oil content of some algae (from Chisti, Biotechnology Advances 25 (2007) 294-306)

Microalga	Oil content (% dry wt)
Botryococcus braunii	25-75
Chlorella sp.	28-32
Crypthecodinium cohnii	20
Cylindrotheca sp.	16-37
Dunaliella primolecta	23
Isochrysis sp.	25-33
Monallanthus salina	>20
Nannochloris sp.	20-35
Nannochloropsis sp.	31-68
Neochloris oleoabundans	35-54
Nitzschia sp.	45-47
Phaeodactylum tricornutum	20-30
Schizochytrium sp.	50-77
Tetraselmis sueica	15-23

The advantages of microalgae over land plants have been summarized in the EPOBIO report (Micro- and macro-algae: utility for industrial application, September 2007, Editor: Dianna Bowles). Both plants (crops) cultivable on arable lands and microalgae grown in open ponds or in confined reactors are potential sources of TAG and fatty acids for industrial purposes and biofuels (Dismukes et al., Current Opinion in Biotechnology 2008, 19:235-240). However, serious concerns have been raised by the intensive agricultural practices the use of crops implies and the diversion of crops from food to non-food chains. Efforts are thus needed to develop novel generation biofuels based on photosynthetic microorganisms.
The main advantages of microalgae in relation to plants for the production of TAG are the following:

This bioresource does not compete with the agro-resources used for animal or human nutrition.
Algal growth can be monitored in controlled and confined conditions in an environmental friendly process using recycled inorganic and organic wastes generated by other human activities and the use of microalgae allows trapping and converting industrial byproduct gases (e.g. CO₂) into valuable organic molecules (Chisti, Biotechnology Advances 25 (2007) 294-306; Chisti, Trends in Biotechnology, 2008, Vol 26, No. 3; Chisti, Journal of Biotechnology 167 (2013) 201-214).
The algal biomass productivity is high, microalgae showing a very high potential of productivity with cost savings when compared to land plants (see Table 2). Their yield is variable and determined by the culturing approach employed: it is relatively low in open pond systems while it can be significantly increased in closed photobioreactors where culture parameters can be controlled.
This bioresource does not depend on a geographical location, or on a season.

Table 2: Comparison of biomass productivity of major crops ("C3" or "C4" type photosynthesis) and microalgae (extract from the « EPOBIO project » report, University of York (09/2007), Table 4).

	Microalgae	"C₄" crops (sorghum, maize, sugarcane...)	"C₃" crops (wheat, sunflower...)
Maximal productivity (T.ha^-1.y^-1)
Microalgae (photobioreactors)	130 to 150	-	-
Higher plants (maximum productivity)	-	72	30
Average productivity in production systems (T.ha^-1.y^-1)
Microalgae (large scale)	10 to 50	-	-
Higher plants (field)	-	10 to 30	8 to 18
Biomass production costs (USD.kg^-1)	0.4 - 40	0.04	0.04

The economic viability of this sector of bioindustry is challenged by the current limitation to combine the overall biomass yield, i.e. dry weight of algal organic matter produced per liter and the proportion of valuable molecules, i.e. sufficiently high proportion of TAG per dry weight for industrial extraction and processing (Chisti, Biotechnology Advances 25 (2007) 294-306; Chisti, Trends in Biotechnology, 2008, Vol 26, No. 3; Chisti, Journal of Biotechnology 167 (2013) 201-214).
In particular, the lipid composition of microalgae is compatible with biodiesel production (Dismukes et al., Current Opinion in Biotechnology 2008, 19:235-240; Scott et al., Current Opinion in Biotechnology 2010, 21:277-286). The rationale for producing biodiesel from microalgae is to use sunlight to convert water and carbon dioxide into biomass. This biomass is then specifically redirected towards the synthesis of oil for the generation biofuels, by applying external stimuli like nutrient stresses, and/or by genetic engineering of metabolism (Dorval Courchesne et al., Journal of Biotechnology 141 (2009) 31-41).
The three most important classes of micro-algae in terms of abundance are the diatoms (Bacillariophyceae), the green algae (Chlorophyceae), and the golden algae (Chrysophyceae) (EPOBIO definition). Diatoms are a major phylum of the phytoplankton biodiversity in oceans, fresh water and various soil habitats. They are responsible for up to 25% of the global primary productivity. Study of this group of eukaryotes has benefited from recent developments on Phaeodactylum tricornutum, a model of pennate diatoms. Diatoms, like other microalgae, are considered a plausible alternative source of hydrocarbons to replace fossil fuels or chemicals from petrochemistry, with the advantage of having a neutral CO₂ balance, based on the hypotheses that CO₂ and water can be efficiently converted into biomass by photosynthesis and that the carbon metabolism could be controlled so that they accumulate energetically-rich TAG. Different phytoplanktonic organisms of the Chromalevolata superphylum have focused the attention for their ability to accumulate TAG, with promising initial yields and appropriate robustness and physical properties to be implemented in an industrial process, including Phaeodactylum tricornutum. Phaeodactylum tricornutum is currently used for the industrial production of omega-3 polyunsaturated fatty acids but industrial implementation for this application and for other applications such as biofuels is still limited by the growth retardation and low yield in biomass when TAG accumulation is triggered using conventional nutrient starvation approaches, such as nitrogen starvation (Chisti, Journal of Biotechnology 167 (2013) 201-214). These approaches have an important drawback which is the limitation of growth that the nitrogen starvation induces. Phaeodactylum tricornutum exhibits interesting properties for an industrial implementation, like the ability to grow in the absence of silicon or the sedimentation of cells that could be useful for harvesting techniques. Attempts to promote TAG accumulation can rely on various strategies that can be combined, including the stimulation of fatty acid and TAG biosynthesis, the blocking of pathways that diverts carbon to alternative metabolic routes and eventually the arrest of TAG catabolism. Small molecules could act on each of these three aspects of TAG metabolism.
It is possible to promote the accumulation of oil in microorganisms by inhibiting or blocking metabolic pathways that direct the carbon fluxes to alternative metabolites. For instance, it is well known that blocking the accumulation of carbohydrate in storage sugars such as starch, promotes the accumulation of oils (Siaut et al., BMC Biotechnology 2011, 11:7).
However, there remains a need for alternative methods to trigger the accumulation of oil when algae are grown in a nitrogen-rich medium, and by trying to avoid blocking carbohydrate storage metabolism. Indeed, the carbohydrates produced after CO₂ photosynthetic conversion serve as a source of carbon for all other organic molecules within the cell, so blocking their storage has a very strong negative impact on cell growth. Other metabolic pathways using carbon might be blocked and allow a redirection of carbon metabolism towards TAG metabolism.
In this aim, the Inventors have now identified that the metabolism of sterols is an alternative sink of carbon, its inhibition in microalgae triggering the accumulation of oils.
Therefore, a first subject of the invention is a method for triggering TAG accumulation in microalgae by inhibiting the sterol metabolism, preferably by inhibiting the synthesis of the sterols, which overcomes the disadvantages listed above.
Within the framework of the invention, the term microalgae refers to microalgae for eukaryotes.
Also in the sense of the present invention, the TAG is built by esterification of a 3-carbon glycerol backbone at positions 1, 2 and 3 by fatty acids. Below, TAG is synthesized by esterification of a glycerol backbone by three fatty acids (R₁, R₂, R₃).
In the sense of the present invention:

Alkyl groups are chosen among (C₁-C₂₆)alkyl groups, preferably (C₁-C₁₈)alkyl groups, and more preferably (C₁-C₆)alkyl groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl and isobutyl radicals;
Alkenyl groups are chosen among hydrocarbon chains of 2 to 26 carbon atoms, preferably 2 to 18, and more preferably 1 to 6, having at least one carbon-carbon double bond. Examples of alkenyl groups include ethenyl, propenyl, isopropenyl, 2,4-pentadienyl;
Alkynyl groups are chosen among hydrocarbon chains of 2 to 26 carbon atoms, preferably 2 to 18, and more preferably 1 to 6, having at least one carbon-carbon triple bond;
Alkynylalkenyl means any group derived from an alkenyl group as defined above wherein a hydrogen atom is replaced by an alkynyl group;
Cycloalkyl groups refer to a monovalent cyclic hydrocarbon radical preferably of 3 to 7 ring carbons. The cycloalkyl group can have one or more double bonds and can optionally be substituted. The term "cycloalkyl" includes, for examples, cyclopropyl, cyclohexyl, cyclohexenyl and the like;
Heteroalkyl groups mean alkyl groups as defined above in which one or more hydrogen atoms to any carbon of the alkyl is replaced by a heteroatom selected from the group consisting of N, O, P, B, S, Si, Sb, Al, Sn, As, Se and Ge. The bond between the carbon atom and the heteroatom may be a single or a double bond. Suitable heteroalkyl groups include cyano, benzoyl, methoxy, acetamide, borates, sulfones, sulfates, thianes, phosphates, phosphonates, and the like;
Alkoxy groups are chosen among (C₁-C₂₀)alkoxy groups, and preferably (C₁-C₄)alkoxy groups such as methyloxy, ethyloxy, n-propyloxy, iso-propyloxy, n-butyloxy, sec-butyloxy, tert-butyloxy and isobutyloxy radicals;
Aryl groups means any functional group or substituent derived from at least one simple aromatic ring; an aromatic ring corresponding to any planar cyclic compound having a delocalized π system in which each atom of the ring comprises a p-orbital, said p-orbitals overlapping themselves. More specifically, the term aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, 2-naphthyl, anthracyl, pyrenyl, and the substituted forms thereof;
Heteroaryl groups means any functional group or substituent derived from at least one aromatic ring as defined above and containing at least one heteroatom selected from P, S, O and N. The term heteroaryl includes, but is not limited to, furan, pyridine, pyrrole, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, tetrazole, pyridazole, pyridine, pyrazine, pyrimidine, pyridazine, benzofurane, isobenzofurane, indole, isoindole, benzothiophene, benzo[c]thiophene, benzimidazole, indazole, benzoxazole, benzisoxazole, benzothiazole, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline, purine and acridine. The aryl and heteroaryl groups of the invention comprise preferably 1 to 12 carbon atoms, and more preferably 5 or 6 carbon atoms;
Arylalkyl means any group derived from an alkyl group as defined above wherein a hydrogen atom is replaced by an aryl or a heteroaryl group;
Arylalkenyl means any group derived from an alkenyl group as defined above wherein a hydrogen atom is replaced by an aryl or a heteroaryl group;
Arylalkynyl means any group derived from an alkynyl group as defined above wherein a hydrogen atom is replaced by an aryl or a heteroaryl group;
Alkylaryl means any group derived from an aryl group as defined above wherein a hydrogen atom is replaced by an alkyl group.

According to the invention, halogen atoms are chosen among bromine, chlorine, fluorine and iodine, and preferably bromine, chlorine and fluorine.
The acid addition salts of the inhibitor of sterol metabolism according to the invention may be for example chosen among hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or hydrogenophosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate, methanesulphonate, benzene-sulphonate and paratoluenesulphonate.
According to a preferred embodiment, in the method of the invention the inhibition of the sterol metabolism is realized by incubating the microalgae with an inhibitor of sterol metabolism, said incubation step being preferably implemented in a nitrogen medium.
The concentration of the inhibitor of sterol metabolism may range from 1 µM to 1M, and preferably from 5 to 20 µM. The incubation step lasts preferably from 24 to 72 hours.
The microalgae of the invention is advantageously selected from microalgae of the diatom phylum, the Chromalveolata phylum, and the Archaeplastidae phylum. Preferably, the microalgae is selected from the diatom micro-algae species Phaeodactylum tricornutum and Thalassiosira pseudonana, the Chromalveolata micro-algae species Nannochloropsis, and more preferably Nannochloropsis gaditana, Nannochloropsis oceanica, Nannochloropsis salina, and the Archaeplastidae micro-algae species Chlamydomonas, Ostreococcus, Chlorella.
Sterol inhibitors consist of molecules that have an effect on any protein activity in the biosynthesis of sterols, a pathway that starts with the biosynthesis of HMG-CoA reductase, followed by the biosynthesis of mevalonic acid, epoxysqualene and then all steroid structures deriving from epoxysqualene (see Figure 1 ). The identification of compounds which inhibit sterol metabolism, preferably with at least a decrease of 20% of total sterol content per cell, can be achieved simply by colorimetric or fluorometric dosage methods such as that commercialized by CellBioLabs (Total Sterol Assay Kit, Colorimetric method, reference STA-384 or Fluorometric method, reference STA-390), based on a treatment by cholesterol oxidase/esterase, which has proved to be efficient on detecting plant sterols (D. Kritchevsky and S. A. Tepper, Clinical Chemistry, Vol. 25, No. 8, 1464-1465 (1979)), or also according to methods such as those exemplified in the applications WO 2010/046385 and WO 97/03202 .
According to an embodiment of the invention, the inhibitor of sterol metabolism is a compound of formula (I) or a salt thereof:
wherein:

n₁ ranges from 1 to 12, and preferably n₁ = 2,
R₁₁ represents a hydrogen atom; a -COR_1a group, in which R_1a is a group selected from a linear or branched alkyl chain, optionally substituted with one or more groups independently selected from hydroxyl, halogen, nitro, optionally substituted benzyl groups, or optionally substituted aryl groups such as phenyl groups, said aryl groups being eventually substituted with one or more groups independently selected from halogen atoms and methyl groups;
R₁₂, R₁₃ and R₁₄, identical or different, represent a hydrogen atom; a linear or branched alkyl chain; a hydroxyl group; a -CH₂OH group; a -COOR_1b group, in which R_1b represents a hydrogen atom, or a linear or branched alkyl chain; a -OSiR_1cR_1dR_1e group, in which R_1c, R_1d and R_1e, identical or different, represent a hydrogen atom, or a linear or branched alkyl chain; or R₁₂ and R₁₃ are fused together to form an exo methylene group;
R₁₅ represents a hydroxyl group; a -OSiR_1cR_1dR_1e group as defined above; a -COOR_1f group, in which R_1f represents a hydrogen atom, or a linear or branched alkyl chain; a-OCOR_1g group, in which R_1g represents a linear or branched alkyl chain, preferably a C₁-C₃ alkyl chain, and more preferably a C₁ alkyl group; or R₁₅ is a carbon forming an ethylenic unsaturation with the tetrahydropyranone ring.

According to a preferred embodiment, R₁₁ is a -COR_1a group, in which R_1a is a linear or branched alkyl chain, and preferably R₁₁ is a -COCH(CH₃)C₂H₅ or -COC(CH₃)₂C₂H₅ group.
Advantageously, R₁₄ represent a C₁-C₆ alkyl chain, preferably a -CH₃ group, and both R₁₂ and R₁₃ represent hydrogen atoms. Alternatively, R₁₂ and R₁₄ may represent a C₁-C₆ alkyl chain, preferably a -CH₃ group, and R₁₃ a hydrogen atom.
According to another preferred embodiment,
n₁ = 2, and R₁₅ represents a hydroxyl group.
According to another embodiment of the invention, the inhibitor of sterol metabolism is a compound of formula (II) or a salt thereof:
wherein:

W₂, X₂, Y₂ and Z₂, identical or different, represent carbon or nitrogen atoms;
R₂₁ represents a hydrogen atom; a linear or branched alkyl chain; a -COR_2a or -S(O)₂-C₆H₅-R_2a group, in which R_2a represents a linear or branched alkyl chain, or an aryl group; a-S(O)₂-R_2b group, in which R_2b represents a linear or branched alkyl chain, or an aryl group, optionally substituted with one or more groups independently selected from linear or branched alkyl chain;
a group of formula:
in which R_2c represents a hydrogen atom or a group selected from a linear or branched alkyl chain, -COR_2a, -COOR_2a; and
R₂₂ and R₂₃, identical or different, represent hydrogen or halogen atoms, and preferably chlorine atoms.

According to a preferred embodiment, X₂ represents a nitrogen atom, and W₂, Y₂ and Z₂ represent carbon atom.
Advantageously, R₂₁ represents a group of formula:
in which R_2c represents a -COR_2a group.
According to another embodiment of the invention, the inhibitor of sterol metabolism is a compound of formula (III) or a salt thereof:
wherein:

W₃, X₃, Y₃ and Z₃ represent carbon, sulphur, nitrogen or oxygen atom, and preferably W₃, X₃, Y₃ and Z₃ represent carbon atoms;
n₃ and n_3', independently, are integer equal to 0 or 1, and preferably n₃ and n_3' are equal to 1;
R₃₁ and R₃₂, identical or different, represent a hydrogen atom, linear or branched alkyl, alkenyl, alkynyl, alkynylalkenyl, cycloalkyl, alkylaryl, arylalkyl, and preferably a benzyl group, arylalkenyl, arylalkynyl, heteroalkyl, heteroaryl groups, or form together a cycloalkyl group comprising 5 to 6 carbon atoms, one or two carbon atoms of said cycloalkyl group being possibly replaced by one or two heteroatoms, preferably nitrogen atoms, or one of R₃₁ or R₃₂ form together with R₃₉ a cycloalkyl group comprising 5 to 6 carbon atoms, one or two carbon atoms of said cycloalkyl group being possibly replaced by one or two heteroatoms, preferably oxygen atoms, said R₃₁ or R₃₂ being optionally substituted with one or more groups independently selected from linear or branched alkyl, cycloalkyl such as
alkynyl, said alkynyl group being preferably a C₅-C₁₂ branched alkynyl group, arylalkyl, aryl, heteroaryl, hydroxyl, halogen, nitro, -COR_3a or -NR_3aR_3b group, in which R_3a and R_3b, identical or different, represent a hydrogen atom or a linear or branched alkyl chain;
R₃₃ represents a hydrogen atom, a linear or branched alkyl chain such as a C₁-C₃ alkyl group, or a nitrile group, and preferably R₃₃ represents a hydrogen atom;
R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ and R₃₉, identical or different, represent hydrogen or halogen atoms, or hydroxyl groups, and preferably R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ and R₃₉ are hydrogen atoms.

The dotted lines of formula (III) represent single or double bonds. Specifically, said W₃-X₃ bond or Y₃-Z₃ bond is single bond respectively when one of W₃ or X₃ of the W₃-X₃ bond, or one of Y₃ or Z₃ of the Y₃-Z₃ bond, is sulphur or oxygen. Said W₃-X₃ bond or Y₃-Z₃ bond is double bond respectively when W₃ and X₃, or Y₃ and Z₃ are carbon or nitrogen.
According to a preferred embodiment, R₃₁ and R₃₂, identical or different, represent C₁-C₆ linear or branched alkyl chains, or an arylalkyl group, optionally substituted with one or more groups independently selected from linear or branched alkyl. Advantageously, R₃₁ represents a C₁-C₆ linear or branched alkyl chain, and R₃₂ a benzyl group substituted with a C₁-C₆ linear or branched alkyl chain, or an alkenyl group substituted with a linear or branched alkynyl group, such as a -(CH₂)n_3"-CH=CH-C≡C-C(CH₃)₃ group in which n_3" ranges from 1 to 6.
According to another embodiment of the invention, the inhibitor of sterol metabolism is a compound of formula (IV) or a salt thereof:

wherein:

R₄₁ and R₄₂, identical or different, represent a hydrogen atom, alkyl, alkenyl, alkynyl, or hydroxyl, -COR_4a or -COOR_4a group, in which R_4a represents a hydrogen atom, linear or branched alkyl, aryl, heteroaryl group, optionally substituted with one or more groups independently selected from alkyl or cycloalkyl groups, preferably C₄-C₆ cycloalkyl groups;
R₄₃ represents a hydrogen atom, or an alkyl group, preferably C₁-C₃ alkyl group; and
R₄₄, R₄₅ and R₄₆, identical or different, represent a hydrogen atom, alkyl, alkoxy, hydroxyl group, or an oxygen atom attached by a double bond, said alkyl group being optionally substituted with one or more halogen atoms, and including optionally in its chain one or more sulfoxide functions.

Advantageously, R₄₁ and R₄₂, identical or different, represent a hydrogen atom, a nitrile, hydroxyl, C₁-C₂ alkyl or -COOR_4a group in which R_4a is in C₁-C₇ and is optionally substituted by a C₄-C₆ cycloalkyl group.
According a preferred embodiment of the invention, the inhibitor of sterol metabolism is selected from Ethinylestradiol, Mevastatin, Simvastatin, Ketoconazole, Butenafine, Terbinafine, Estrone.
The inhibitors of sterol metabolism of the invention are all acting on the "mevalonate" pathway, and not on the "non-mevalonate" pathway of synthesis of sterols (see Figure 1 ).
More specifically:

the inhibitors of sterol metabolism of formula (I) are acting as HMG-CoA reductase inhibitors (Liu et al., Mol. Biol. Rep. (2010) 37:1391-1395),
the inhibitors of sterol metabolism of formula (II) are acting as Cytochrome P450c17 inhibitors (Berman et al., Molecular and Biochemical Parasitology, 20 (1986) 85-92),
the inhibitors of sterol metabolism of formula (III) are acting as squalene epoxidase inhibitors (Belter et al., Biol. Chem., Vol. 392, 1053-1075 (2011)), and
the inhibitors of sterol metabolism of formula (IV) are acting as steroid structural analogs interacting with protein involved in sterol metabolism including Estrone (Merola and Arnold, Science, Vol. 144, 301-302 (1964)) and Ethinylestradiol (Koopen et al., Journal of Lipid Research, Vol. 40,1999).

Another subject-matter of the invention is a method for producing fatty acids comprising a triggering of triacylglycerols accumulation step in microalgae as defined according to the invention.
The invention also relates to a method for producing biofuels comprising a triggering of triacylglycerols accumulation step in microalgae as defined according to the invention, and a subsequent trans-esterification step of the obtained triacylglycerols, as described by Zhang et al., Bioresource Technology 147 (2013) 59-64.
The invention also concerns a method for producing pharmaceutical or cosmetic compositions comprising a triggering of triacylglycerols accumulation step in microalgae as defined according to the invention.
The invention also relates to a method for producing human food and animal feed supplements comprising a triggering of triacylglycerols accumulation step in microalgae as defined according to the invention.
The methods of the invention may comprise one or more extraction steps after the triggering of triacylglycerols accumulation step in microalgae. The extraction step may be implemented using solvents or another extraction method well known form the skilled artisan.
Another subject-matter of the invention relates to the use of an inhibitor of sterol metabolism to accumulate triglycerides in microorganism, preferably in microalgae, more preferably in microalgae of the diatom phylum, and still more preferably in the diatom microalgae species Phaeodactylum tricornutum.
Advantageously, the invention concerns the use of an inhibitor of sterol metabolism selected from the compounds of formula (I), (II), (III), and (IV), such as Ethinylestradiol, Mevastatin, Simvastatin, Ketoconazole, Butenafine, Terbinafine, Estrone, to accumulate triglycerides in microorganism, preferably in microalgae, more preferably in microalgae of the diatom phylum, and still more preferably in the diatom microalgae species Phaeodactylum tricornutum. The invention also concerns the use of combination of inhibitors of sterol metabolism selected from the compounds of formula (I), (II), (III), and (IV), such as Ethinylestradiol, Mevastatin, Simvastatin, Ketoconazole, Butenafine, Terbinafine, Estrone, as well as their combination with other methods known to enhance the accumulation of TAG in microalgae, in particular a shortage of nutrient, and more preferably a shortage of nitrogen.
In addition to the above provisions, the invention also comprises other provisions which will emerge from the remainder of the description which follows, and also to the appended drawings in which:

Figure 1 gives a schematic view of the sterol biosynthetic pathway via mevalonic acid, and represents the action of Mevastatin and Simvastatin (Liu et al., Mol. Biol. Rep. (2010) 37:1391-1395), Estrone (Merola and Arnold, Science, Vol. 144, 301-302 (1964), Butenafine and Terbinafine (Belter et al., Biol. Chem., Vol. 392, 1053-1075 (2011)), and Ketoconazole (Berman et al., Molecular and Biochemical Parasitology, 20 (1986) 85-92),
Figure 2 represents pictures of a Phaeodactylum cell visualized by confocal microscopy after a 72h cultivation in Nitrogen-rich N(+) or in Nitrogen-starved N(-) medium On the left side (A and C), cells are shown in phase contrast ("phase"). On the right side (B and D), cells are shown after excitation of Nile red at 488 nm and emission at 519 nm,
Figure 3 a-j illustrates the dose response for Ethinylestradiol, Mevastatin, Simvastatin and Estrone, and
Figure 4 illustrates the dose response for Ethinylestradiol, Mevastatin and Simvastatin with an evaluation of the TAG content by staining with Nile Red and an evaluation of cells by counting in an aliquote fraction using a Malassez cell.

EXAMPLES:

1) Materials & Methods

1) 1-Phaeodactylum tricornutum strain and growth conditions

Phaeodactylum tricornutum (Pt1) Bohlin Strain 8.6 CCMP2561 (Culture Collection of Marine Phytoplankton, now known as NCMA: National Center for Marine Algae and Microbiota) was used in experiments.
Pt1 was grown at 20°C in 250 mL flask using "enriched artificial seawater" (ESAW) medium, prepared following the recommendations of the Canadian Center for the Culture of Microorganisms.

To prepare the ESAW medium, four separated solutions are prepared, two solutions of salts (solutions 1 and 2), one solution of nutrients, and one solution of vitamins. Salts are added in order to distilled deionized water (DDW). When the salts in solutions 1 and 2 are completely dissolved, the solutions 1 and 2 are mixed together. The total volume is diluted with DDW.

Table 3: Compositions of the ESAW salts solutions 1 and 2

Solution 1: Anhydrous salts	Molecular weight (g.mol^-1)	Amount to weight (g/L solution)	Concentration (mM)
NaCl	58.44	20.756	362.7
Na₂SO₄	142.04	3.477	25.0
KCl	74.56	0.587	8.03
NaHCO₃	84	0.17	2.067
KBr	119.01	0.0845	0.725
H₃BO₃	61.83	0.022	0.372
NaF	41.99	0.0027	0.0657

Solution 2: Hydrated salts
MgCl₂.6H₂O	203.33	9.395	47.18
CaCl₂.2H₂O	147.03	1.316	9.134
SrCl₂.6H₂O	266.64	0.0214	0.082

Table 4: Nutrient Enrichment Stocks

Solutions		Stock concentration (g.L^-1)	Final concentration (µM)
1	NaNO₃	46.67	549.1
2*	Na₂ glycerophosphate	6.67	21.8
3	Na₂SiO₃.9H₂O	15.00	105.6
4**	Na₂EDTA.2H₂O	3.64	9.81
	Fe(NH₄)₂(SO₄)₂.6H₂O***	2.34	5.97
	FeCl₃.6H₂O	0.16	0.592
5	MnSO₄.4H₂O	0.54	2.42
	ZnSO₄.7H₂O	0.073	0.254
	CoSO₄.7H₂O	0.016	0.0569
	Na₂MoO₄.2H₂O	0.126	0.520
	Na₂EDTA.2H₂O	1.89	5.05
6	H₃BO₃	3.80	61.46
7	NaSeO₃	0.00173	0.001

* Na₂ glycerophosphate can be replaced with an equimolar stock of Na₂HPO₄.
** Na₂EDTA.2H₂O is added before the trace metals Fe(NH₄)₂(SO₄)₂.6H₂O and FeCl₃.6H₂O.
*** Fe(NH₄)₂(SO₄)₂.6H₂O can be replaced with an equimolar stock of FeCl₃.
Solution 5 is adjusted to pH = 6 with 2 g of Na₂CO₃.
Solution 4 can be heated to dissolve the iron.

Table 5: Vitamin Stocks

Vitamin Stock	Stock concentration (g.L^-1)	Final concentration (mM)
Thiamine	0.1	2.97 × 10^-1
Vitamin B12	0.002	1.47 × 10^-3
Biotin	0.001	4.09 × 10^-3

To prepare the ESAW medium, the solutions are filtered through 0.45 µm membrane filter with a glass fiber prefilter. A flask is acid-washed in 10% HCl and rinsed in distilled water before first use. To 1 L of filtered salt solution, 1 mL of Nutrient Enrichment Stock solutions 1, 2, 4, 5, 6 and 7, 2 mL of Nutrient Enrichment Stock solution 3, and 2 mL of the Vitamin Stock are added (Tables 4 and 5). To reduce precipitation during autoclaving, 1.44 mL of 1N HCl and 0.12g of sodium bicarbonate are added. The obtained ESAW medium is then sterilized by autoclaving.
Cells were grown on a 12:12 light (450 µEinstein - 1 sec^-1) / dark cycle (an Einstein defined the energy in one mole (6.022 × 10²³) of photons). Cells were sub-cultured every week by inoculate fresh media with 1/5 of previous culture. Nitrogen-rich N(+) medium contained no source of nitrogen. Nitrogen-starved N(-), medium contained 0.05 g/L NaNO₃. To monitor cell growth, a genetically modified strain containing a Histone H4 protein fused to the yellow fluorescent protein was used (Siaut et al., Gene 406 (2007) 23-35).

1) 2- Principle of Nile Red staining of oil droplets

Accumulation of oil droplets can be monitored by Nile Red (Sigma Aldrich) fluorescent staining (Excitation wavelength at 485 nm; emission at 525 nm) as described by Ren et al. (Biotechnology for Biofuels 2013, 6:143). Cells were diluted and adjusted to a cell density that was linearly correlated with Nile Red fluorescence. Nile Red solution (40 µL of 2.5 µg.mL^-1 stock concentration, in 100% DMSO) was added to 160 µL cell suspension. Specific fluorescence was determined by dividing Nile Red fluorescence intensity by the number of cells. Oil bodies stained with Nile Red were then visualized using a Zeiss AxioScope.A1 microscope (FITC filter; Excitation wavelength at 488 nm; emission at 519 nm).
Lipid droplets can be visualized. In Figure 2 , a Phaeodactylum cell was visualized by confocal microscopy after a 72h cultivation in Nitrogen-rich N(+) medium or in Nitrogen-starved N(-) medium. On the left side, cells are shown in phase contrast ("phase"). On the right side, cells are shown after excitation of Nile red at 488 nm and emission at 519 nm. Lipid droplets can be clearly visualized.
This principle was use to measure the presence of oil in Phaeodactylum tricornutum simply by using a spectrofluorometer (in these conditions, to lower the detection of other fluorophores within the cell, such as chlorophylls, excitation was at 530 nm and emission at 580 nm).

1) 3- Alternative method for oil level detection

Alternatively, oil is extracted using solvents or another extraction method, separated and purified by thin layer chromatography and methanolyzed to produce fatty acid methyl esters and quantified by gas chromatography coupled to a ionization flame detector or a mass spectrometer.
TAG were extracted from 200 mg of freeze-dried Phaeodactylum tricornutum cells in order to prevent lipid degradation. Briefly, cells were frozen in liquid nitrogen immediately after harvest. The freeze-dried cell pellet was resuspended in 4 mL of boiling ethanol for 5 minutes followed by the addition of 2 mL of methanol and 8 mL of chloroform at room temperature. The mixture was then saturated with argon and stirred for 1h at room temperature. After filtration through glass wool, cell remains were rinsed with 3 mL of chloroform/methanol (2:1, v/v). In order to initiate biphase formation, 5 mL of NaCl 1% was then added to the filtrate. The chloroform phase was dried under argon before re-solubilization of the lipid extract in pure chloroform. To isolate TAG, lipids were run on silica gel thin layer chromatography (TLC) plates (Merck) with hexane/diethylether/acetic acid (70:30:1, v/v). Lipids were then visualized under UV light after pulverization of 8-anilino-1-naphthalenesulfonic acid at 2% in methanol. They were then scraped off from the TLC plates for further analyses. For acyl profiling and quantification of TAG, fatty acids were methylated using 3 mL of 2.5% H₂SO₄ in methanol during 1h at 100°C (including standard amounts of 21:0). The reaction was stopped by the addition of 3 mL of water and 3 mL of hexane. The hexane phase was analyzed by gas liquid chromatography (Perkin Elmer) on a BPX70 (SGE) column. Methylated fatty acids were identified by comparison of their retention times with those of standards and quantified by surface peak method using 21:0 for calibration. Extraction and quantification were done at least 3 times.

1) 4- Principle of cell normalization

The number of cells in a sample can be evaluated using a fluorescent reporter, like the strain by Siaut et al. (Gene 406 (2007) 23-35) containing a genetic construction with Histone H4 protein fused to the Yellow Fluorescent Protein (YFP) (Figure 5A of Siaut et al., Gene 406 (2007) 23-35).
For cell counting, we can either use this strain called "ptYFP" and measure the fluorescence emitted by the YFP at 530 nm after excitation at 515 nm, or use any strain and estimate cell numbers by counting with a Malassez grid (supplier: Mareinfeld).

1) 5- Incubation of Phaeodactylum tricornutum with inhibitors of the sterol metabolism and detection of oil accumulation triggered by the treatment

On the first day, we prepared 48 well plates by adding 4 mm glass beads sterilized with Ultra-Violet exposure in each well.
We prepared fresh suspensions of microalgae in exponential growth phase. For cell normalization based on YFP fluorescence, cells of Phaeodactylum tricornutum containing a YFP reporter (ptYFP) cultured in N(+) ESAW medium were centrifuged at 3,500 rpm, 5 min. For cell normalization based on counting using a Malassez grid, cells of the Pt1 strain of Phaeodactylum tricornutum cultured in N(+) ESAW medium were centrifuged at 3,500 rpm, 5 min. The supernatant was discarded and the pellet suspended in N(-) ESAW. The microalgae were then centrifuged at 3,500 rpm, 5 min. The supernatant was discarded and the pellet suspended in N(-) ESAW. Cells were then diluted to 1x10⁶ cells/mL in N(-) ESAW. Samples were then separated into two batches. One was supplemented with 1 µL/mL of 46.7 g/L NaNO₃ stock to obtain a suspension of cells in N(+) ESAW medium. Another was left without NaNO₃ to obtain a N(-) ESAW culture as a control for high lipid accumulation. In a 48-well clear NUNC plate, 450 µL/well of 1x10⁶ cells/mL were dispensed.
For a dose-response analysis, each well of the 48-well plate was then subjected to an appropriate incubation with 0, 1, 10 or 100 µM of inhibitor of sterol metabolism using 50 µL of the following: 50 µL of a 10 time concentrated solution of inhibitor of sterol metabolism (5% DMSO:95% N(+) ESAW) or 50 µL of Nitrogen-rich medium without any inhibitor of sterol metabolism (5% DMSO:95% N(+) ESAW) or Nitrogen-starved medium without any inhibitor of sterol metabolism (5% DMSO:95% N(-) ESAW) as a positive control.
For an analysis of the effect of a single dose, an incubation was performed with 0, and a chosen concentration of inhibitor of sterol metabolism (50 µM).
In all cases, the edge of the plate was sealed using a parafilm. Plates were incubated for 48h in an incubator with top lighting, 20°C, 100 rpm, 12h/12h light/dark.
After an incubation of 48 hours, fluorescence was measured at the following excitation/emission wavelengths, 530/580 nm (to evaluate a baseline fluorescence prior Nile Red addition) and 515/530 nm (to evaluate YFP fluorescence). Following this first measure, 40 µL of Nile Red (2.5 µg/mL stock concentration, in 100% DMSO) are added. Plates are mixed and incubated 20 minutes at room temperature, protected from light. Nile Red fluorescence is then measured using a spectrofluorometer (excitation 530 nm/emission 580 nm).

1) 6- Inhibitors of sterol metabolism

Inhibitors of sterol metabolism were obtained from the Prestwick library for their ability to trigger the accumulation of lipid droplets within the cells of Phaeodactylum tricornutum, and then purchased from Sigma-Aldrich.

Table 6: Inhibitors of sterol metabolism selected from the Prestwick library

Chemical name	Structure	Molecular Formula
Ketoconazole		C₂₆H₂₈C₁₂N₄O₄
Mevastatin		C₂₃H₃₄O₅
Butenafine		C₂₃H₂₇N
Simvastatin		C₂₅H₃₈O₅
Estrone		C₁₈H₂₂O₂
Ethinylestradiol		C₂₀H₂₄O₂

2) Results

Phaeodactylum tricornutum was incubated for 48h in presence of 10 µM of Ketoconazole, Mevastatin, Butenafine, Simvastatin, Estrone, Ethinylestradiol and Terbinafine.
In all cases the presence of oil per cell increased by a factor of at least 1.5, based on Nile Red staining.
Figure 3a-j illustrates the dose response for Ethinylestradiol, Mevastatin, Simvastatin and Estrone with an evaluation of the TAG content by staining with Nile Red, and an evaluation of cells by monitoring YFP fluorescence from an expressed Histone H4 reporter gene. On the left, Figure 3 a-j shows the Nile Red levels in percent of untreated cells and the number of cells estimated by the YFP fluorescence expressed in percent of untreated cells. The right panels show the increase of oil content per cell, by the ratio of Nile Red fluorescence/YFP fluorescence, expressed in percent of untreated cells. The oil content per cell increases with drug concentration and consequently level of sterol metabolism inhibition and ranges from 120 to 400 % when compared to untreated cells.
Figure 4 illustrates the dose response for Ethinylestradiol (A), Mevastatin (B) and Mevastatin (C) with an evaluation of the TAG content by staining with Nile Red and an evaluation of cells by counting in an aliquote fraction using a Malassez cell. In histograms of Figure 4 , the white bars indicate the evaluation of cell numbers in percent of untreated cells, and the black bars indicate the Nile Red per 10⁶ cell, in percent of untreated cells. The oil content per cell, and consequently level of sterol metabolism inhibition, increases with incubation of 50 µM of inhibitor, and ranges from 120 to 350% when compared to untreated cells.

Claims

A method for triggering triacylglycerols accumulation in microalgae by inhibiting the sterol metabolism.
A method according to Claim 1, wherein the inhibition of the sterol metabolism is realized by incubating the microalgae with an inhibitor of sterol metabolism.
A method according to Claim 2, wherein the incubation step is implemented in a nitrogen medium.
A method according to Claims 1 to 3, wherein the microalgae is selected from microalgae of the diatom phylum, the Chromalveolata phylum, and the Archaeplastidae phylum.
A method according to Claim 4, wherein the microalgae is selected from the diatom micro-algae species Phaeodactylum tricornutum and Thalassiosira pseudonana, the Chromalveolata micro-algae species Nannochloropsis, and preferably Nannochloropsis gaditana, Nannochloropsis oceanica, Nannochloropsis salina, and the Archaeplastidae micro-algae species Chlamydomonas, Ostreococcus, Chlorella.
A method according to Claims 1 to 5, wherein the inhibitor of sterol metabolism is a compound of formula (I) or a salt thereof:
wherein:
- n₁ ranges from 1 to 12,

- R₁₁ represents a hydrogen atom; a -COR_1a group, in which R_1a is a group selected from a linear or branched alkyl chain, optionally substituted with one or more groups independently selected from hydroxyl, halogen, nitro, optionally substituted benzyl groups, or optionally substituted aryl groups such as phenyl groups, said aryl groups being eventually substituted with one or more groups independently selected from halogen atoms and methyl groups;

- R₁₂, R₁₃ and R₁₄, identical or different, represent a hydrogen atom; a linear or branched alkyl chain; a hydroxyl group; a -CH₂OH group; a -COOR_1b group, in which R_1b represents a hydrogen atom, or a linear or branched alkyl chain; a -OSiR_1cR_1dR_1e group, in which R_1c, R_1d and R_1e, identical or different, represent a hydrogen atom, or a linear or branched alkyl chain; or R₁₂ and R₁₃ form together an exo methylene group;

- R₁₅ represents a hydroxyl; a -OSiR_1cR_1dR_1e group; a -COOR_1f group, in which R_1f represents a hydrogen atom, or a linear or branched alkyl chain; a -OCOR_1g group, in which R_1g represents a linear or branched alkyl chain; or R₁₅ is a carbon forming an ethylenic unsaturation with the tetrahydropyranone ring.
A method according to Claim 1 to 5, wherein the inhibitor of sterol metabolism is a compound of formula (II) or a salt thereof:
wherein:
- W₂, X₂, Y₂ and Z₂, identical or different, represent carbon or nitrogen atoms;

- R₂₁ represents a hydrogen atom; a linear or branched alkyl chain; a -COR_2a or -S(O)₂-C₆H₅-R_2a group, in which R_2a represents a linear or branched alkyl chain, or an aryl group; a - S(O)₂-R_2b group, in which R_2b represents a linear or branched alkyl chain, or an aryl group, optionally substituted with one or more groups independently selected from linear or branched alkyl chain;
a group of formula:
in which R_2c represents a hydrogen atom, or a group selected from a linear or branched alkyl chain, -COR_2a, -COOR_2a; and

- R₂₂ and R₂₃, identical or different, represent hydrogen or halogen atoms.
A method according to Claims 1 to 5, wherein the inhibitor of sterol metabolism is a compound of formula (III):
wherein:
- W₃, X₃, Y₃ and Z₃ represent carbon, sulphur, nitrogen or oxygen atom;

- n₃ and n_3', independently, are integer equal to 0 or 1, and preferably n₃ and n_3' are equal to 1;

- R₃₁ and R₃₂, identical or different, represent a hydrogen atom, linear or branched alkyl, alkenyl, alkynyl, alkynylalkenyl, cycloalkyl, alkylaryl, arylalkyl, arylalkenyl, arylalkynyl, heteroalkyl, heteroaryl groups, or form together a cycloalkyl group comprising 5 to 6 carbon atoms, one or two carbon atoms of said cycloalkyl group being possibly replaced by one or two heteroatoms, or one of R₃₁ or R₃₂ form together with R₃₉ a cycloalkyl group comprising 5 to 6 carbon atoms, one or two carbon atoms of said cycloalkyl group being possibly replaced by one or two heteroatoms, said R₃₁ or R₃₂ being optionally substituted with one or more groups independently selected from linear or branched alkyl, cycloalkyl such as
alkynyl, arylalkyl, aryl, heteroaryl, hydroxyl, halogen, nitro, -COR_3a or -NR_3aR_3b group, in which R_3a and R_3b, identical or different, represent a hydrogen atom, or a linear or branched alkyl chain;

- R₃₃ represents a hydrogen atom, a linear or branched alkyl chain, or a nitrile group;

- R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ and R₃₉, identical or different, represent hydrogen or halogen atoms, or hydroxyl groups.
A method according to Claims 1 to 5, wherein the inhibitor of sterol metabolism is a compound of formula (VI):
wherein:
- R₄₁ and R₄₂, identical or different, represent a hydrogen atom, alkyl, alkenyl, alkynyl, or hydroxyl, -COR_4a or -COOR_4a group, in which R_4a represents a hydrogen atom, a linear or branched alkyl, aryl, heteroaryl group, optionally substituted with one or more groups independently selected from alkyl or cycloalkyl groups;

- R₄₃ represents a hydrogen atom, or an alkyl group; and

- R₄₄, R₄₅ and R₄₆, identical or different, represent a hydrogen atom, an alkyl, alkoxy, hydroxyl group, or an oxygen atom attached by a double bond, said alkyl group being optionally substituted with one or more halogen atoms, and including optionally in its chain one or more sulfoxide functions.
A method according to Claims 1 to 9, wherein the inhibitor of sterol metabolism is selected from Ethinylestradiol, Mevastatin, Simvastatin, Ketoconazole, Butenafine, Terbinafine, Estrone.
A method for producing fatty acids comprising a triggering of triacylglycerols accumulation step in microalgae as defined according to Claims 1 to 10.
A method for producing biofuels comprising a triggering of triacylglycerols accumulation step in microalgae as defined according to Claims 1 to 10, and a subsequent trans-esterification step of the obtained triacylglycerols.
A method for producing pharmaceutical or cosmetic compositions comprising a triggering of triacylglycerols accumulation step in microalgae as defined according to Claims 1 to 10.
A method for producing food supplements comprising a triggering of triacylglycerols accumulation step in microalgae as defined according to Claims 1 to 10.
The use of an inhibitor of sterol metabolism to accumulate triglycerides in microorganism, and preferably in microalgae.
The use according to Claim 15, wherein the inhibitor of sterol metabolism is chosen from the compounds of formula (I), (II), (III), and (IV), as defined respectively is Claims 6, 7, 8 and 9, or combination thereof.
The use according to Claim 16, wherein the inhibitor of sterol metabolism is chosen from Ethinylestradiol, Mevastatin, Simvastatin, Ketoconazole, Butenafine, Terbinafine, Estrone, or combination thereof.